Cytotoxic pyridoacridine alkaloids

ABSTRACT

Shermilamine D, a novel cytotoxic pyridoacridine alkaloid of Formula 1,                    
     has been isolated from the tunicate  Cystodytes violatinctus.  The structure of the compound has been established on the basis of 1-D and 2-D NMR data.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention is a Section 371 filing of PCT/GB98/03282 filedNov. 3, 1998, which designated the United States. The PCT applicationwas published in the English language on May 14, 1999. Priority wasclaimed from Application No. 9,723206.0 filed Nov. 3, 1997 in GreatBritain.

The present invention relates to new cytotoxic pyridoacridine alkaloids,one of which, Shermilamine D, isolated from the tunicate Cystodytesviolatinctus.

BACKGROUND OF THE INVENTION

Marine organisms, especially soft cerals, sponges and tunicates, providemany secondary metabolites and exhibit a varying degree of biologicalactivity (Reference 13: Faulkner, D. J. Nat.Prod.Reports., 1997, 14,259-302 and references cited therein). An important family of thesemetabolites is the alkaloid family; in 1983 it was reported thestructure of a marine alkaloid, amphimedine (Reference 14: Schmitz, F.J. et al. J.Am.Chem.Soc. 1983, 105, 4835-4836), which was the first of anew class of marine-derived alkaloids that collectively have come to beknown as the “pyridoacridines”. Since then, over 40 additional exampleshave been published (Reference 1: Molinski, T. F. Chem.Rev. 1993, 93,1825-1838). The pyridoacridines are highly colored marine derivedalkaloids majority based on the 11H-pyrido[4,3,2-mn]acridine skeleton,as shown below:

A family of remarkable cytotoxic marine pyridoacridine alkaloids is theShermilamine family (Reference 6: Scheuer, P. J. et al. J.Org.Chem.1988, 53, 4619-4620. Reference 5: Scheuer, P. J. et al. J.Org.Chem.1989, 54, 4231-4232. Reference 3: Kashman, Y. et al. J.Org.Chem. 1989,54, 5335-5337. Reference 12: Barrows, L. R.; Ireland, C. M. et al,J.Med.Chem. 1994, 37, 3819-3827), which structure is showed below:

Shermilamines were all isolated from tunicates, A and B from Trididemnumsp. and C from Cystodytes sp.

SUMMARY OF THE INVENTION

The present invention provides new pyridoacridines having the followingformula (I):

wherein R is independently selected from the group consisting ofhydrogen or bromine.

More particularly, the present invention relates to Shermilamine D (R═Hin formula (I)), extracted and isolated from the tunicate Cystodytesviolatinctus.

Shermilamine D exhibits antitumor activity. In particular, ShermilamineD exhibits antitumor activity against cell lines derived from humansolid tumors, such as human lung carcinoma, human colon carcinoma andhuman melanoma, and, the like, it is active against other tumor celllines, like leukemia and lymphoma.

The present invention also provides a method of testing a mammalaffected by a malignant tumor sensitive to a compound with the formula(I), which comprises administering a therapeutically effective amount ofthe compound with the formula (I), or a pharmaceutical compositionthereof.

The present invention further provides pharmaceutical compositions whichcontain as active ingredient a compound with the formula (I), as well asprocess for its preparation.

A further aspect of the invention is a method for preparing the compoundShermilamine D (R═H in the formula (I)), which comprises extraction andisolation from the tunicate Cystodytes violatinctus.

PREFERRED EMBODIMENTS OF THE INVENTION

Examples of pharmaceutical compositions include any solid (tablets,pills, capsules, granules, etc.) or liquid (solutions, suspensions oremulsions) with suitable formulation of oral, topical or parenteraladministration, and they may contain the pure compound or in combinationwith any carrier or other pharmacologically active compounds. Thesecompositions may need to be sterile when administered parenterally.

The correct dosage of a pharmaceutical composition comprising compoundswith the formula (I), will vary according to the pharmaceuticalformulation, the mode of application, and the particular situs, host andtumor being treated. Other factors like age, body weight, sex, diet,time of administration, rate of excretion, condition of the host, drugcombinations, reaction sensitivities and severity of the disease shallbe taken into account. Administration can be carried out continuously orperiodically within the maximum tolerated dose.

ANTITUMOUR ACTIVITY

Cells were maintained in logarithmic phase of growth in Eagle's MinimumEssential Medium, with Earle's Balanced Salts, with 2.0 mM L-glutamine,with non-essential amino acids, without sodium bicarbonate (EMEM/neaa);supplemented with 10% Fetal Calf Serum (FCS), 10⁻² M sodium bicarbonateand 0.1 g/l penicillin+G streptomycin sulfate.

A screening procedure has been carried out to determine and compare theantitumor activity of these compounds, using an adapted form of themethod described by Bergeron et al. (Reference 15: Raymond J. Bergeron,Paul F. Cavanaugh, Jr., Steven J. Kline, Robert G. Hughes, Jr., Gary T.Elliot and Carl W. Porter. Antineoplastic and antiherpetic activity ofspermidine catecholamide iron chelators. Biochem. Bioph. Res. Comm.1984, 121, 848-854). The antitumor cells employed were P-388 (suspensionculture of a lymphoid neoplasm from DBA/2 mouse), A-549 (monolayerculture of a human lung carcinoma), HT-29 (monolayer culture of a humancolon carcinoma) and MEL-28 (monolayer culture of a human melanoma).

P-388 cells were seeded into 16 mm wells at 1×10⁴ cells per well in 1 mlaliquots of MEM 5FCS containing the indicated concentration of drug. Aseparate set of cultures without drug was seeded as control growth toensure that cells remained in exponential phase of growth. Alldeterminations were carried out in duplicate. After three days ofincubation at 37° C., 10% CO₂ in a 98% humid atmosphere, anapproximately IC₅₀ was determined by comparing the growth in wells withdrug to the growth in wells control.

A-549, HT-29 cells were seeded into 16 mm wells at 2×10⁴ cells per wellin 1 ml aliquots of MEM 10FCS containing the indicated concentration ofdrug. A separate set of cultures without drug was seeded as controlgrowth to ensure that cells remained in exponential phase of growth. Alldeterminations were carried out in duplicate. After three days ofincubation at 37° C., 10% CO₂ in a 98% humid atmosphere, the wells werestained with 0.1% Crystal Violet. An approximately IC₅₀ was determinedby comparing the growth in wells with drug to the growth in wellscontrol.

The results were given in the following table:

IC₅₀ (μM) P-388 A-549 HT-29 MEL-28 Shermilamine D 1.33 0.27 2.66 0.53

EXTRACTION AND ISOLATION

Low-resolution mass spectra were recorded on a EIMS mass spectrometer. Hand ¹³C-NMR spectra were recorded on a Bruker ARX-500 spectrometer. Allchemical shifts were reported with respect to TMS (δ=0 ppm).

The tunicate Cystodytes violatinctus was collected by SCUBA at 5 m depthon Prevoyante reef in lagoon of Mayotte (Comoros islands), north-west ofMadagascar during April 1996. Reference samples (AM-35) are deposited atthe Museum National d'Histoire Naturelle of Paris. The freshly collectedanimals was immediately frozen at −25° C. The freeze-dried animal (600 gweight after extraction) were homogenized and extracted withmethanol-chloroform 1:2 (500 ml×4) at room temperature. The extractswere evaporated into vacuto to give an aqueous phase wich was extractedwith chloroform. Evaporation of the chloroform extracts afforded an oilyresidue (1.6 g). The crude extract is partitioned between 20% aq. MeOHand CCL₄, the organic extracts were evaporated and the residue is thenchromatographed, first on Sephadex LH-20 (hexane, CHCl₃, MeOH, 2:1:1)and then MeOH-washed-silica-gel chromatography starting with CHCl₃ andadding MeOH, 1-10%. The compound Shermilamine D (2% from the crudeextract) comes out with 5% MeOH in CHCl₃. Shermilamine D is a foamingoil, m/z 376 (C₂,H₂₀N₄OS).

For NMR data see next Table:

NMR data for Shermilamine D C No. δ_(C)(m)^(a) δ_(H)(m, J in Hz)^(a,b)HMBC to C No. 2 150.7(d) 8.40(d, 4.8) 3,3a,13b 3 106.8(d) 7.20(d, 4.7)2,8b 3a 140.2(s) — — 3b 116.3(s) — — 4 123.7(d) 7.80(d, 7.9) 3b,6,7a 5120.6(d) 6.98(t, 7.5) 3a,3b,6,7 6 131.7(d) 7.33(bt, 7.5) 4,7a 7 116.4(d)6.84(d, 8.1) 3b,5 7a 140.7(s) — — 8a 132.9(s) — — 8b 116.7(s) — — 9109.6(s) — — 9a 121.4(s) — — 11  29.7(t) 3.50(bs) 9a,12 12 163.5(a) — —13a 121.3(s) — — 13b 137.3(s) — — 14  27.4(t) 2.96(bt, 4.0)8a,9a,15,17,18 15  58.7(t) 2.62(bt, 4.8) 14,17,18 17  44.9(q) 2.50(s)15,18 18  44.9(q) 2.50(s) 15,17 NH-8 — 10.2(bs) — NH-13 — 9.05(bs)9a.11.13b aNMR solvent = CDCl₃; 500 MHz for ¹H, 125 MHz for ¹³C. bNMRsolvent = CDCl₃—CD₃OD (4:1).

REFERENCES

1. Molinski T. F. Chem.Rev. 1993, 93, 1825-1838.

2. Scheuer, P. J. et al, J.Org.Chem. 1990, 55, 4426-4431.

3. Kashman, Y. et al, J.Org.Chem. 1989, 54, 5335-5337.

4. Schmitz, F. J.; Helm, D. v. d. J.Org.Chem. 1991, 56, 804-808.

5. Scheuer, P. J. et al. J.Org.Chem. 1989, 54, 4231-4232.

6. Scheuer, P. J. et al. J.Org.Chem. 1988, 53, 4619-4620.

7. Steffan, B. et al. Tetrahedron, 1993, 49, 6223-6228.

8. Spector, I. et al. J.Cell.Physiol. 1993, 157, 481-492.

9. Burres, N. S. et al. Cancer Res. 1989, 49, 5267-5274.

10. Ciufolini, M. A. et al. J.Am.Chem.Soc. 1995, 117, 12460-12469.

11. Taraporewala, I. B. et al. J.Med.Chem. 1992, 35, 2744-2752.

12. Barrows, L. R.; Ireland, C. M. et al. J.Med.Chem. 1994, 37,3819-3827.

13. Faulkner, D. J.Nat.Prod.Reports, 1974, 14, 259-302.

14. Schmitz, F. J. et al. J.Am.Chem.Soc. 1983, 105, 4825-4836.

15. Raymond J. Bergeron, Paul F. Cavanaugh, Jr, Steven J. Kline, RobertG. Hughes, Jr., Gary T. Elliot and Carl W. Porter, Antineoplastic andantiherpetic activity of spermidine catechloamide iron chelators.Biochem. Bioph. Res. Comm. 1984, 121, 848-854.

What is claimed is:
 1. A compound having the following formula (I):

wherein R is independently selected from the group consisting ofhydrogen or bromine.
 2. A compound, Shermilamine D, according to claim1, having the following formula:


3. A method of treating a mammal affected by a malignant tumor sensitiveto a compound with the formula (I), as defined in claim 1, whichcomprises administering to the affected individual a therapeuticallyeffective amount of the compound or a pharmaceutical compositionthereof.
 4. A method of treating a mammal affected by a malignant tumorsensitive to Shermilamine D, as defined in claim 2, which comprisesadministering to the affected individual a therapeutically effectiveamount of Shermilamine D or a pharmaceutical composition thereof.
 5. Apharmaceutical preparation which contains as active ingredient acompound with the formula (I), as defined in claim
 1. 6. Apharmaceutical preparation which contains as active ingredientShermilamine D, as defined in claim
 2. 7. (Amended) A method of treatinga mammal affected by a malignant tumor which comprises administering tothe affected individual a therapeutically effective amount of a compoundwith the formula (I), as defined in claim 1, together with one or moreother antitumoral compounds.
 8. (Amended) A method of treating a mammalaffected by a malignant tumor which comprises administering to theaffected individual a therapeutically effective amount of ShermilamineD, as defined in claim 2, together with one or more other antitumoralcompounds.
 9. A method for preparing Shermilamine D, as defined in claim2, which comprises extraction and isolation from the tunicate Cystodytesviolatinctus.